Phase II study of nivolumab in patients with genetic alterations in DNA damage repair and response who progressed after standard treatment for metastatic solid cancers (KM-06).

BACKGROUND: Immune-modulating antibodies targeting programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) have demonstrated promising antitumor efficacy in various types of cancers, especially highly mutated ones. Genetic alterations in DNA damage response and repair (DDR) genes can lead to genetic instability, often accompanied by a high tumor mutation burden (TMB). However, few studies have validated the aberration of DDR genes as a predictive biomarker for response to immune-modulating antibodies. METHODS: The KM-06 open-label, multicenter, single-arm, phase II trial evaluated the safety and efficacy of nivolumab in refractory solid cancers with DDR gene mutations assessed by clinically targeted sequencing. Nivolumab (3 mg/kg) was administered every 2 weeks until disease progression, unacceptable toxicity, or for 24 months. The primary endpoint was the objective response rate (ORR) as per RECIST V.1.1 criteria. RESULTS: A total of 48 patients were enrolled in the study (median age 61, 58.3% male). The most common cancer type was colorectal cancer (41.7%), followed by prostate and biliary tract cancer (8.3% each). Eight patients achieved a partial response as their best overall response, resulting in an ORR of 17.8%. The disease control rate was 60.0%. The median progression-free survival was 2.9 months. Treatment-related adverse events of any grade and grade ≥3 occurred in 44 (91.7%) and 4 (8.3%) patients, respectively. Clinically targeted sequencing data inferred both TMB and microsatellite instability (MSI). Using a TMB cut-off of 12 mut/Mb, there were significant differences in overall survival (p=0.00035), progression-free survival (p=0.0061), and the best overall response (p=0.05). In the RNA sequencing analysis, nivolumab responders showed activation of the interleukin signaling pathway. Patients who experienced early progression presented high epithelial-mesenchymal transition signaling pathway activation. The responders exhibited a marked increase in PD-1-/Ki67+CD8 T cells at the early stage of treatment (C3D1) compared with non-responders (p=0.03). CONCLUSIONS: In this phase II trial, nivolumab demonstrated moderate efficacy and manageable toxicity in patients with solid cancer harboring DDR gene mutations. A high TMB (>12 mut/Mb) and MSI score (>2.5) determined through clinically target sequencing presented significant discriminatory power for the nivolumab response. TRIAL REGISTRATION NUMBER: NCT04761744.

Usefulness of The Rapid Antigen Test in Detecting SARS-CoV-2 for Infection Control in Hospitals.

BACKGROUND: We aimed at evaluating the diagnostic performance of rapid antigen test (RAT) compared to PCR for severe acute respiratory syndrome coronavirus 2 and the possible transmission of infection to close contacts from patients with negative RAT and positive polymerase chain reaction (PCR) results. MATERIALS AND METHODS: Patients/guardians urgently requiring admission to the ward on the same day had been hospitalized with RAT-negative result before the PCR results were available. We performed an epidemiologic investigation of the close contacts of those with negative RAT but positive PCR results after hospitalization. RESULTS: A total of 4,237 RATs were performed from March to August 2022. When the PCR test was used as the reference, RAT had a sensitivity of 28.8% (17/59; 95% confidence interval [CI]: 17.8 - 42.1), a specificity of 100% (4,220/4,220; 95% CI: 99.9 - 100.0), a positive predictive value of 100.0% (17/17; 95% CI: 100.0 - 100.0), and a negative predictive value of 99.0% (4,178/4,220; 95% CI: 99.3 - 99.8). The epidemiologic investigation revealed that among the 32 patients with negative RAT and subsequent positive PCR results after admission into multi-patient room, two (6.3%) showed secondary coronavirus disease 2019. CONCLUSION: The secondary transmission rate from patients with negative RAT and positive PCR results was low. Our data suggest that RAT may be useful for rapid exclusion of high transmissible cases. However, further evaluation using whole genome sequencing is needed to determine the potential for transmissibility in cases showing a negative RAT but a positive PCR result.

Causes of a Low Measles Seroprevalence among Young Healthcare Workers in Korea.

BACKGROUND: Sporadic measles outbreaks have continued to occur in Korea, mainly in adults in their 20s and 30s, most notably in 2014 and 2019. We here evaluated the possible causes of a low seroprevalance of measles by testing young healthcare workers (HCWs). MATERIALS AND METHODS: This study was conducted in a 2,743-bed tertiary-care hospital in Seoul between 2020 and 2021. We performed a measles antibody test (chemiluminescence immunoassay), measured the IgM/IgG index ratio, and conducted an avidity test at 1-month after Measles-Mumps-Rubella (MMR) vaccination in HCWs who had been seronegative for measles. Measles vaccination histories were obtained from the national vaccine registry. RESULTS: Of the 3,173 HCWs newly employed in our hospital during the study period, 54 with a negative measles IgG at commencement were enrolled. Thirty six (67%) of these subjects were female, and the median age was 25 years (interquartile range [IQR]: 24 - 27). Fourty nine (91%) showed seroconversion at 1 month after the first vaccination. Of these individuals, 38 received both measles IgM and IgG test, and all had an IgM/IgG index <1. Of the 49 seroconverters, all HCWs showed a high avidity index. According to the national immunization registry, 45 (83%) received at least 2 doses of an MMR vaccination. CONCLUSION: Secondary vaccine failure may underlie vaccine failure in young Korean adults. HCWs born after 1985 with a negative measles antibody may need only a single dose booster vaccination rather than a 2-dose vaccination regimen.

Method to Determine the Optimal Aptamer-to-Bead Ratio by Using Flow Cytometry.

Research on the effective attachment of aptamers to beads, which is essential for using aptamers, has made relatively little progress. Here, we demonstrate a new method based on flow cytometry to determine the optimal aptamer-to-bead ratio for aptamer immobilization. The fluorescence intensity increased with a gradual two-fold increase in the aptamer fluorescence concentration, peaked at an aptamer-to-bead ratio of 2.56 × 105, and tended to decrease at higher ratios. A similar pattern was observed in an additional analysis using fluorescence microscopy. However, measurement of the free aptamer concentration after the aptamer-bead conjugation reaction revealed a large aptamer loss compared to the 1.28 × 105 aptamer-bead ratio. In addition, the binding efficiency of the aptamer/bead to the target was highest at the aptamer-to-bead ratio of 1.28 × 105. Taken together, our data suggest that the proposed method is the best and easiest for determining the optimal aptamer-to-bead ratio.

Active surveillance testing to reduce transmission of carbapenem-resistant, gram-negative bacteria in intensive care units: a pragmatic, randomized cross-over trial.

BACKGROUND: In intensive care unit (ICU) settings, the transmission risk of carbapenem-resistant, gram-negative bacteria (CRGNB) is high. There is a paucity of data regarding the effectiveness of interventions, including active screening, preemptive isolation, and contact precautions, to reduce transmission of CRGNB. METHODS: We conducted a pragmatic, cluster-randomized, non-blinded cross-over study in 6 adult ICUs in a tertiary care center in Seoul, South Korea. ICUs were randomly assigned to perform active surveillance testing with preemptive isolation and contact precautions (intervention) or standard precautions (control) during the initial 6-month study period, followed by a 1-month washout period. During a subsequent 6-month period, departments that used standard precautions switched to using interventional precautions and vice versa. The incidence rates of CRGNB were compared between the two periods using Poisson regression analysis. RESULTS: During the study period, there were 2268 and 2224 ICU admissions during the intervention and control periods, respectively. Because a carbapenemase-producing Enterobacterales outbreak occurred in a surgical ICU (SICU), we excluded admissions to the SICU during both the intervention and control periods and performed a modified intention-to-treat (mITT) analysis. In mITT analysis, a total of 1314 patients were included. The acquisition rate of CRGNB was 1.75 cases per 1000 person-days during the intervention period versus 3.33 cases per 1000 person-days during the control period (IRR, 0.53 [95% confidence interval (CI) 0.23-1.11]; P = 0.07). CONCLUSIONS: Although this study was underpowered and showed borderline significance, active surveillance testing and preemptive isolation could be considered in settings with high baseline prevalence of CRGNB. Trial registration Clinicaltrials.gov Identifier: NCT03980197.

Erratum: Correction of Funding in the Article "Rates of COVID-19 Infection Among Healthcare Workers in Designated COVID-19 Wards and General Wards".

This corrects the article on p. e308 in vol. 37, PMID: 36345254.

Rates of COVID-19 Infection Among Healthcare Workers in Designated COVID-19 Wards and General Wards.

BACKGROUND: To evaluate the effects of isolating coronavirus disease 2019 (COVID-19) patients in general wards, we compared the rates of COVID-19 infection in nurses and nursing assistants working in COVID-19 designated wards and in general wards of our hospital from 1 October 2021 to 21 April 2022. METHODS: This study was conducted in a 2,700-bed tertiary care hospital in Seoul, Korea. Designated wards comprised single, negative pressure rooms and a 100% outdoor air system. RESULTS: During the study period, a total of 2,698 nurses and nursing assistants were employed at our hospital, of whom 310 (11%) were working in the designated wards, and the remaining 2,388 (89%) in the general wards, and among whom 1,158 (43%) were diagnosed with COVID-19. The healthcare workers (HCWs) in the designated wards were less frequently diagnosed with COVID-19 than those in the general wards (31% vs. 45%, P < 0.001). During the period before patients with COVID-19 were isolated in general wards, and during the period after these cases were isolated in general ward, HCWs in designated wards were less frequently infected with the virus than those in general wards (7% vs. 11%, P = 0.039; and 23% vs. 33%, P < 0.001, respectively). CONCLUSION: HCWs in designated wards have a lower rate of contracting COVID-19 than those in general wards. A lack of exposure to undiagnosed cases and their caregivers, greater care with social distancing outside the hospital, higher rates of 3-dose vaccinations, and the use of isolation rooms with negative pressure may be associated with this finding.

Evaluation of In-Hospital Cluster of COVID-19 Associated With a Patient With Prolonged Viral Shedding Using Whole-Genome Sequencing.

BACKGROUND: Patients with hematologic malignancies may produce replication-competent virus beyond 20 days of SARS-CoV-2 infection. However, data regarding the transmission of SARS-CoV-2 from patients with prolonged viral shedding is limited. METHODS: In May 2022, four additional cases of COVID-19 were reported in a hematologic ward at a tertiary care hospital in South Korea, after an 8-week isolation of a patient with prolonged viral shedding. We performed whole-genome sequencing (WGS) of SARS-CoV-2 to evaluate the possibility of post-isolation transmission from this prolonged viral shedding. RESULTS: A patient (case 1) with acute myeloid leukemia was released from isolation 54 days after the diagnosis of COVID-19 based on rising Ct value of up to 29.3, and moved to a six-patient room. On days 10 and 11 post-isolation, his doctor (case 2) and 2 patients who were his roommates (case 3, 4) had positive SARS-CoV-2 PCR results. Additionally, 16 days post-isolation, another patient (case 5) in a remote room had positive SARS-CoV-2 PCR result. All the three patients were hospitalized for ≥ 14 days when they were diagnosed with SARS-CoV-2 infection. Except for case 3, the remaining 4 cases were available for WGS, which revealed that case 1 exhibited a 7 nucleotides difference in comparison to cases 4 and 5 and case 2 displayed a 20 nucleotides difference compared with case 1, while sequences of cases 4 and 5 were identical. CONCLUSIONS: Despite the possibility of transmission from the patient with prolonged viral shedding, no evidence of the transmission of SARS-CoV-2 from the patient with prolonged positive RT-PCR using WGS was found.

Usefulness of universal SARS-CoV-2 PCR testing for inpatients and their caregivers during the omicron era.

Background: Since January 2022, the Omicron variant has become the dominant strain in South Korea, and COVID-19 cases among hospitalized patients and their guardians or caregivers have increased. We evaluated the usefulness of universal periodic screening for SARS-CoV-2 in patients and resident caregivers in a South Korean tertiary care hospital. Methods: We evaluated the reason for testing in COVID-19 confirmed patients and resident caregivers during their hospitalization from March 3 to 31, 2022. The rate of positive PCR universal testing in hospital (or residency) (HD) on days 3 and 7 in asymptomatic patients and caregivers were evaluated. The test for SARS-CoV-2 was done by RT-PCR. Results: During the study period, 677 patients were diagnosed with COVID-19. The reasons for testing were the symptoms (226 (33%)), pre-admission test (183 (27%)), exposure to COVID-19 (124 (18%)), universal testing on HD 3 (94 (14%)), and that on HD 7 (34 (5%)). Caregivers (n = 340) were tested during their residency due to exposure to COVID-19 cases, 103 (30%); universal testing on HD 3, 90 (26%); symptom development, 46 (14%); pre-stay, 39 (11%); and universal testing on HD 7, 29 (9%). The positive test rates of inpatients and caregivers on HD 3 and HD 7 were as follows: 1.4% (93/6553) and 2.1% (55/2646) in inpatients, and 1.3% (79/5989) and 1.7% (35/2106) in caregivers, respectively. Conclusions: Universal testing, regardless of symptom or epidemiologic link, is useful for detecting pre-symptomatic and asymptomatic COVID-19 cases among hospitalized patients and caregivers and preventing a nosocomial outbreak during the Omicron era.

Nosocomial Outbreak by Delta Variant From a Fully Vaccinated Patient.

BACKGROUND: The potential for a nosocomial outbreak of coronavirus disease 2019 (COVID-19) from a fully vaccinated individual is largely unknown. METHODS: In October 2021, during the time when the delta variant was dominant, a nosocomial outbreak of COVID-19 occurred in two wards in a tertiary care hospital in Seoul, Korea. We performed airflow investigations and whole-genome sequencing (WGS) of the virus. RESULTS: The index patient developed symptoms 1 day after admission, and was diagnosed with COVID-19 on day 4 post-admission. He was fully vaccinated (ChAdOx1 nCoV-19) 2 months before the diagnosis. Three inpatients and a caregiver in the same room, two inpatients in an adjacent room, two inpatients in rooms remote from the index room, and one nurse on the ward tested positive. Also, two resident doctors who stayed in an on-call room located on the same ward tested positive (although they had no close contact), as well as a caregiver who stayed on an adjacent ward, and a healthcare worker who had casual contact with this caregiver. Samples from five individuals were available for WGS, and all showed ≤ 1 single-nucleotide polymorphism difference. CCTV footage showed that the index case walked frequently in the corridors of two wards. An airflow study showed that the air from the corridor flowed into the resident on-call room, driven by an air circulator that was always turned on. CONCLUSION: Transmission of severe acute respiratory syndrome coronavirus 2 from a fully vaccinated index occurred rapidly via the wards and on-call room. Care must be taken to not use equipment that can change the airflow.

Transmission and Infectious SARS-CoV-2 Shedding Kinetics in Vaccinated and Unvaccinated Individuals.

Importance: Data are limited on whether patients with breakthrough COVID-19 infection have the potential to significantly contribute to the spread of SARS-CoV-2. Objective: To compare the secondary attack rate and infectious viral shedding kinetics of SARS-CoV-2 between fully vaccinated individuals (breakthrough infection group) and partially or unvaccinated individuals (nonbreakthrough infection group). Design, Setting, and Participants: This cohort study assessed secondary transmission by analyzing the epidemiologic data of health care workers, inpatients, and caregivers diagnosed with COVID-19 during hospitalization or residence in a tertiary care hospital between March 1, 2020, and November 6, 2021. To evaluate viral shedding kinetics, the genomic RNA of SARS-CoV-2 was measured using polymerase chain reaction and performed virus culture from daily saliva samples of individuals with mild COVID-19 infected with the Delta variant who were isolated in a community facility in Seoul, South Korea, between July 20 and August 20, 2021. Exposures: COVID-19 vaccination. Main Outcomes and Measures: The secondary attack rate and infectious viral shedding kinetics according to COVID-19 vaccination status. Results: A total of 173 individuals (median [IQR] age, 47 [32-59] years; 100 female [58%]) with COVID-19 were included in the secondary transmission study, of whom 50 (29%) had a breakthrough infection. Secondary transmission was significantly less common in the breakthrough infection group than in the nonbreakthrough infection group (3 of 43 [7%] vs 29 of 110 [26%]; P = .008). In the viral shedding kinetics study, 45 patients (median age, 37 years [IQR, 25-49 years]; 14 female [31%]) infected with the Delta variant were included, of whom 6 (13%) were fully vaccinated and 39 (87%) were partially or unvaccinated. Although the initial genomic viral load was comparable between the 2 groups, viable virus in cell culture was detected for a notably longer duration in partially vaccinated (8 days after symptom onset) or unvaccinated (10 days after symptom onset) individuals compared with fully vaccinated individuals (4 days after symptom onset). Conclusions and Relevance: In this cohort study, although the initial genomic viral load was similar between vaccinated and unvaccinated individuals, fully vaccinated individuals had a shorter duration of viable viral shedding and a lower secondary attack rate than partially vaccinated or unvaccinated individuals. Data from this study provide important evidence that despite the possibility of breakthrough infections, COVID-19 vaccinations remain critically useful for controlling the spread of SARS-CoV-2.

Discovery of Reversible Covalent Bruton's Tyrosine Kinase Inhibitors PRN473 and PRN1008 (Rilzabrutinib).

Bruton's tyrosine kinase (BTK), a Tec family tyrosine kinase, is critical in immune pathways as an essential intracellular signaling element, participating in both adaptive and immune responses. Currently approved BTK inhibitors are irreversible covalent inhibitors and limited to oncology indications. Herein, we describe the design of covalent reversible BTK inhibitors and the discoveries of PRN473 (11) and rilzabrutinib (PRN1008, 12). These compounds have exhibited potent and durable inhibition of BTK, in vivo efficacy in rodent arthritis models, and clinical efficacy in canine pemphigus foliaceus. Compound 11 has completed phase 1 trials as a topical agent, and 12 is in phase 3 trials for pemphigus vulgaris and immune thrombocytopenia.

Risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission Among Healthcare Workers Dining in Hospital Staff Cafeterias.

BACKGROUND: Preventive measures are needed to reduce the risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among healthcare workers (HCWs). Notably, hospital staff are usually exposed when they are unmasked. There are limited data on the risk of transmission during mealtimes at hospital staff cafeterias. We aimed to evaluate the risk of transmission in cafeterias. METHODS: From January 2020 through September 2021, we analyzed the risk of SARS-CoV-2 transmission through closed-circuit television and radio-frequency identification tracking and follow-up testing when 33 HCWs, who were eventually diagnosed as coronavirus disease 2019 (COVID-19), ate in staff cafeterias during the infectious period. The seats were arranged so the HCWs would sit on either side without facing each other. There were no plastic barriers installed, and HCWs were encouraged not to talk during meals. RESULTS: Three of the 119 individuals who ate at seats next (about 30 cm) to index during the period of transmission and underwent follow-up SARS-CoV-2 polymerase chain reaction tests were diagnosed with COVID-19 (2.5%; 95% confidence interval, 0.5-7.4%). Among the 98 HCWs who were investigated about talking during meals, there was a higher attack rate among those who spoke with each other than among those who did not (12.5% [3/24] vs. 0% [0/74], P = 0.013). CONCLUSION: The risk of transmission in a hospital's employee cafeterias is not high with side-by-side seating, especially in the absence of conversation.

Comparison of the Clinical Outcomes of Patients With Positive Xpert Carba-R Tests for Carbapenemase-Producing Enterobacterales According to Culture Positivity.

BACKGROUND: We aimed to compare the clinical outcomes of patients with positive Xpert Carba-R assay results for carbapenemase-producing Enterobacterales (CPE) according to CPE culture positivity. METHODS: We retrospectively collected data for patients with positive CPE (positive Xpert Carba-R or culture) who underwent both tests from August 2018 to March 2021 in a 2700-bed tertiary referral hospital in Seoul, South Korea. We compared the clinical outcomes of patients positive for Xpert Carba-R according to whether they were positive (XPCP) or negative (XPCN) for CPE culture. RESULTS: Of 322 patients with CPE who underwent both Xpert Carba-R and culture, 313 (97%) were positive for Xpert Carba-R for CPE. Of these, 87 (28%) were XPCN, and 226 (72%) were XPCP. XPCN patients were less likely to have a history of previous antibiotic use (75.9% vs 90.3%; P = .001) and to have Klebsiella pneumoniae carbapenemase (21.8% vs 48.9%; P < .001). None of the XPCN patients developed infection from colonization within 6 months, whereas 13.4% (29/216) of the XPCP patients did (P < .001). XPCN patients had lower transmission rates than XPCP patients (3.0% [9/305] vs 6.3% [37/592]; P = .03). There was no significant difference in CPE clearance from positive culture results between XPCN and XPCP patients (40.0% [8/20] vs 26.7% [55/206]; P = .21). CONCLUSIONS: Our study suggests that XPCN patients had lower rates of both infection and transmission than XPCP patients. The Xpert Carba-R assay is clinically useful not only for rapid identification of CPE but also for predicting risks of infection and transmission when performed along with culture.

Recurrence of Classic Scabies in a Patient Who had Unchanged Tie String of the T-Cannula.

A 64-year old woman who underwent lung transplantation and had T-cannula contracted classic scabies from her daughter. The patient was treated with 5% permethrin 10 times and had two consecutive negative follow-up microscopic examinations. However, the patient had recurrent symptoms and diagnosed with recurrent classic scabies 22 days after the last treatment. Unchanged tie string and unapplied topical agent around the T-cannula skin lesion may be reasons for recurrence. Thus, accurate application of topical treatment and changing the tie string of the t-cannula is needed to prevent a recurrence.

SP-1154, a novel synthetic TGF-ß inhibitor, alleviates obesity and hepatic steatosis in high-fat diet-induced mice.

OBJECTIVE: Obesity-induced inflamed visceral adipose tissue (VAT) secretes pro-inflammatory cytokines thereby promoting systemic inflammation and insulin resistance which further exacerbate obesity-associated nonalcoholic fatty liver disease (NAFLD). Transforming growth factor (TGF)-ß /Smad3 signaling plays a crucial role in the inflammatory events within the VAT. Here, we investigate whether SP-1154, a novel synthetic verbenone derivative, can inhibit TGF-ß/Smad3 signaling thereby exhibiting a therapeutic effect against obesity-induced inflamed VAT and subsequent NAFLD in high-fat diet-induced mice. METHODS: NAFLD was induced by a high-fat diet (60% fat) for 20 weeks using the male C57BL/6 mice. SP-1154 (50 mg/kg) was orally given daily for 20 weeks. In vivo VAT- and systemic inflammation were measured by using 18F-fluorodeoxyglucose positron emission tomography and C-reactive protein levels. Both insulin tolerance- and glucose tolerance test were performed to assess the status of insulin resistance and glucose intolerance. Histological and molecular analyses were performed on harvested liver and VAT. KEY FINDINGS: SP-1154 inhibited TGF-ß/Smad3 signaling pathway and remarkably suppressed high-fat diet-induced VAT inflammation and its related systemic inflammation. Furthermore, SP-1154 significantly improved insulin sensitivity with glucose homeostasis and reduced hepatic steatosis. SP-1154 significantly improves VAT inflammation and obesity-related NAFLD. CONCLUSION: Our novel findings support the potential use of SP-1154 as a therapeutic drug for obesity and its related NAFLD by targeting the inflamed VAT.

Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1.

Ferroptosis is a type of programmed necrosis triggered by iron-dependent lipid peroxidation. We investigated the role of B-cell translocation gene 1 (BTG1) in cystine and methionine deficiency (CST/Met (-))-mediated cell death. CST/Met (-) depleted reduced and oxidized glutathione in hepatocyte-derived cells, increased prostaglandin-endoperoxide synthase 2 expression, and promoted reactive oxygen species accumulation and lipid peroxidation, as well as necrotic cell death. CST/Met (-)-mediated cell death and lipid peroxidation was specifically inhibited by pretreatment with ferroptosis inhibitors. In parallel with cell death, CST/Met (-) blocked global protein translation and increased the expression of genes associated with the integrated stress response. Moreover, CST/Met (-) significantly induced BTG1 expression. Using a BTG1 promoter-harboring reporter gene and siRNA, activating transcription factor 4 (ATF4) was identified as an essential transcription factor for CST/Met (-)-mediated BTG1 induction. Although knockout of BTG1 in human HAP1 cells did not affect the accumulation of reactive oxygen species induced by CST/Met (-), BTG1 knockout significantly decreased the induction of genes associated with the integrated stress response, and reduced lipid peroxidation and cell death in response to CST/Met (-). The results demonstrate that CST/Met (-) induces ferroptosis by activating ATF4-dependent BTG1 induction.